The present invention concerns methods for diagnosis and treatment of disorders characterized by abnormal cellular signal transduction. The following is a discussion of relevant art, none of which is admitted to be prior art to the invention.
Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of tyrosine residues on proteins. The phosphorylation state of a protein is modified through the reciprocal actions of tyrosine kinases (TKs) and tyrosine phosphatases (TPs).
Receptor tyrosine kinases (RTKs) belong to a family of transmembrane proteins and have been implicated in cellular signaling pathways. The predominant biological activity of some RTKs is the stimulation of cell growth and proliferation, while other RTKs are involved in arresting growth and promoting differentiation. In some instances, a single tyrosine kinase can inhibit, or stimulate, cell proliferation depending on the cellular environment in which it is expressed.
RTKs are composed of at least three domains: an extracellular ligand binding domain, a transmembrane domain and a cytoplasmic catalytic domain that can phosphorylate tyrosine residues. Ligand binding to membrane-bound receptors induces the formation of receptor dimers and allosteric changes that activate the intracellular kinase domains and result in the self-phosphorylation (autophosphorylation and/or transphosphorylation) of the receptor on tyrosine residues. Their intrinsic tyrosine kinase is activated upon ligand binding, thereby initiating a complex signal transduction pathway that begins with receptor autophosphorylation and culminates in the tyrosine phosphorylation of a variety of cellular substrates and ultimately in the initiation of nuclear events necessary for the overall cell response. Individual phosphotyrosine residues of the cytoplasmic domains of receptors may serve as specific binding sites that interact with a host of cytoplasmic signaling molecules, thereby activating various signal transduction pathways.
The intracellular, cytoplasmic, non-receptor protein tyrosine kinases do not contain a hydrophobic transmembrane domain or an extracellular domain and share non-catalytic domains in addition to sharing their catalytic kinase domains. Such non-catalytic domains include the SH2 domains (SRC homology domain 2) and SH3 domains (SRC homology domain 3). The non-catalytic domains are thought to be important in the regulation of protein-protein interactions during signal transduction.
A central feature of signal transduction (for reviews, see Posada and Cooper, Mol. Biol. Cell 3:583–392, 1992; Hardie, Symp. Soc. Exp. Biol. 44:241–255, 1990), is the reversible phosphorylation of certain proteins. Receptor phosphorylation stimulates a physical association of the activated receptor with target molecules. Some of the target molecules such as phospholipase Cγ are in turn phosphorylated and activated. Such phosphorylation transmits a signal to the cytoplasm. Other target molecules are not phosphorylated, but assist in signal transmission by acting as adapter molecules for secondary signal transducer proteins. For example, receptor phosphorylation and the subsequent allosteric changes in the receptor recruit the Grb-2/SOS complex to the catalytic domain of the receptor where its proximity to the membrane allows it to activate ras.
The secondary signal transducer molecules generated by activated receptors result in a signal cascade that regulates cell functions such as cell division or differentiation. Reviews describing intracellular signal transduction include Aaronson, Science, 254:1146–1153, 1991; Schlessinger, Trends Biochem. Sci., 13:443–447, 1988; and Ullrich and Schlessinger, Cell, 61:203–212, 1990.
RTKs are important regulators of developmental processes, as reflected by the high level of tyrosine phosphorylation in the early mouse embryo, which decreases with progressing development and is low in adult animal tissues (Pasquale and Singer, Proc. Natl. Acad. Sci. USA 88:5449–5453, 1989). For example, the mouse c-kit proto-oncogene plays a key role in the migrational behavior of specific cell types in mouse development (Chabot et al., Nature 335:88–89, 1988; Geissler et al., Cell 55:185–192, 1988; Nocka et al., Genes Dev. 3:816–826, 1989).
Disruption of the platelet-derived growth factor receptor α (PDGF-Rα) gene is responsible for the mouse patch mutation, which is characterized by prominent anatomical abnormalities in homozygotes (Stephenson et al., Proc. Natl. Acad. Sci. USA 88:6–10, 1991). Moreover, Flk-1, the cognate receptor for the vascular endothelial growth factor (VEGF), was shown to be a major regulator of vasculogenesis and angiogenesis (Millauer et al., Cell 72:835–846, 1993). Finally, in Drosophila, the RTK sevenless has a well established function in the control of photoreceptor cell fate (Basler and Hafen, Science 243:931–934, 1989), as does the RTK torso in the formation of terminal structures of Drosophila larva (Sprenger et al., Nature 338:478–483, 1989).
Among adult tissues, the brain contains the highest level of protein kinase activity, comparable to that found in embryonic tissues (Maher, P. A., J. Cell. Biol. 112:955–963, 1991). Members of the trk family of RTKs have well documented roles in promoting the differentiation and survival of diverse groups of neurons of the central and peripheral nervous systems (reviewed in Raffioni et al., Annu. Rev. Biochem. 62:823–850, 1993). The eck/eph RTK subfamily (Hirai et al., Science 238:1717–1720, 1987) currently comprises the largest subgroup of RTKs (Sajjadi and Pasquale, Oncogene 8:1807–1813, 1993), with most members being expressed in the developing or adult brain.
While RTKs such as eck (Lindberg and Hunter, Mol. Cell. Biol. 10:6316–6324, 1990), Hek2 (Böhme et al., Oncogene 8:2857–2862, 1993), Cek6, Cek9, and Cek10 (Sajjadi and Pasquale, Oncogene 8:1807–1813, 1993) have been reported to be widely expressed in a variety of tissues, Elk and Cek5 transcripts have been found predominantly in the brain (Letwin et al., Oncogene 3:621–627, 1988; Pasquale et al., J. Neuroscience 12:3956–3967, 1992).
As first noted by Maisonpierre et al. (Maisonpierre et al., Oncogene 8:3277–3288, 1993), there is a subclass of RTKs within the eck/eph family which, while being strongly expressed in the brain, are also found in other tissues, especially during embryogenesis. This subfamily includes Ehk-1, Ehk-2, (Maisonpierre et al., Oncogene 8:3277–3288, 1993), Mek4, Cek4, Hek (Sajjadi et al., New Biol. 3:769–778, 1991; Wicks et al., Proc. Natl. Acad. Sci. USA 89:1611–1615, 1992), eek (Chan and Watt, Oncogene 6:1057–1061, 1991), Sek (Nieto et al., Development 116:1137–1150, 1992; Gilardi-Hebenstreit et al., Oncogene 7:2499–2506, 1992), Cek7 and Cek8 (Sajjadi and Pasquale, Oncogene 8:1807–1813, 1993), whose members are more related to each other than to either of the above-mentioned kinases.